中华烧伤杂志
中華燒傷雜誌
중화소상잡지
16
2009年
3期
197-201
,共5页
沈雁%李孝建%梁蓉%李延仓%张琰%梁佩红%戴丽冰%杨小红%谭见容%李叶扬%黄粤
瀋雁%李孝建%樑蓉%李延倉%張琰%樑珮紅%戴麗冰%楊小紅%譚見容%李葉颺%黃粵
침안%리효건%량용%리연창%장염%량패홍%대려빙%양소홍%담견용%리협양%황월
皮肤%干细胞%组织工程%仿生材料%几丁质
皮膚%榦細胞%組織工程%倣生材料%幾丁質
피부%간세포%조직공정%방생재료%궤정질
Skin%Stem cell%Tissue engineering%Biomimetic materials%Chitin
目的 了解大鼠表皮干细胞(ESC)与几丁质膜共同构建皮肤组织工程覆盖物的可行性.方法 采用冷消化法和Ⅳ型胶原贴壁法分离培养大鼠ESC.倒置显微镜观察细胞生长情况,激光共聚焦显微镜检测其DNA和RNA表达,澳玛蓝比色法测定ESC生长曲线,流式细胞仪检测ESC表面标志物CD29、CD71、CD49d和CD34,免疫组织化学法测定细胞角蛋白15(CK15)、CK19和P63阳性表达.检测不同倍比稀释几丁质膜浸出液对细胞的影响,并设常规培养为对照组;观察ESC在载体上的生长情况.结果 分离培养的细胞经鉴定确认为ESC,细胞倍增时间为48 h.其表面标志物CD29,CD49d为阳性,CD71、CD34为阴性;CK15、CK19、P63为阳性.几丁质膜浸出液培养的细胞与对照组比较,在1:8～1:512等比稀释时,有轻微的促细胞增殖作用但差异无统计学意义(P>0.05).ESC在几丁质膜上培养2～4周时,肉眼可见棋盘样细胞集落,显微镜下见大量ESC在纤维上增殖.结论 几丁质膜可以作为ESC的培养载体,两者有较好的生物相容性.
目的 瞭解大鼠錶皮榦細胞(ESC)與幾丁質膜共同構建皮膚組織工程覆蓋物的可行性.方法 採用冷消化法和Ⅳ型膠原貼壁法分離培養大鼠ESC.倒置顯微鏡觀察細胞生長情況,激光共聚焦顯微鏡檢測其DNA和RNA錶達,澳瑪藍比色法測定ESC生長麯線,流式細胞儀檢測ESC錶麵標誌物CD29、CD71、CD49d和CD34,免疫組織化學法測定細胞角蛋白15(CK15)、CK19和P63暘性錶達.檢測不同倍比稀釋幾丁質膜浸齣液對細胞的影響,併設常規培養為對照組;觀察ESC在載體上的生長情況.結果 分離培養的細胞經鑒定確認為ESC,細胞倍增時間為48 h.其錶麵標誌物CD29,CD49d為暘性,CD71、CD34為陰性;CK15、CK19、P63為暘性.幾丁質膜浸齣液培養的細胞與對照組比較,在1:8～1:512等比稀釋時,有輕微的促細胞增殖作用但差異無統計學意義(P>0.05).ESC在幾丁質膜上培養2～4週時,肉眼可見棋盤樣細胞集落,顯微鏡下見大量ESC在纖維上增殖.結論 幾丁質膜可以作為ESC的培養載體,兩者有較好的生物相容性.
목적 료해대서표피간세포(ESC)여궤정질막공동구건피부조직공정복개물적가행성.방법 채용랭소화법화Ⅳ형효원첩벽법분리배양대서ESC.도치현미경관찰세포생장정황,격광공취초현미경검측기DNA화RNA표체,오마람비색법측정ESC생장곡선,류식세포의검측ESC표면표지물CD29、CD71、CD49d화CD34,면역조직화학법측정세포각단백15(CK15)、CK19화P63양성표체.검측불동배비희석궤정질막침출액대세포적영향,병설상규배양위대조조;관찰ESC재재체상적생장정황.결과 분리배양적세포경감정학인위ESC,세포배증시간위48 h.기표면표지물CD29,CD49d위양성,CD71、CD34위음성;CK15、CK19、P63위양성.궤정질막침출액배양적세포여대조조비교,재1:8～1:512등비희석시,유경미적촉세포증식작용단차이무통계학의의(P>0.05).ESC재궤정질막상배양2～4주시,육안가견기반양세포집락,현미경하견대량ESC재섬유상증식.결론 궤정질막가이작위ESC적배양재체,량자유교호적생물상용성.
Objective To investigate the feasibility of constructing a skin tissue engineering cover-ing on chitinous membrane using rat epidermal stem cells (ESCs). Methods Rat ESCs were isolated and cultured by cold digestive method and collagen type Ⅳ adherent method. Cell colonies were observed with in-verted microscope. Expressions of DNA and RNA of ESCs were detected with laser scanning confocal micro-scope. Growth curves of cells were determined with Alamar BlueTM colorimetric method. Expressions of sur-face markers of ESCs (CD29, CD71, CD49d, and CD34) were detected with flow cytometer. Positive expres-sions of CK15, CK19, and P63 of ESCs were determined by immunohistochemistry. Influence of original chi-tinous membrane leachate in different dilutions on ESCs was observed. Condition of growth of ESCs on the ve-hicle was observed. Results Isolated cultured cells were verified as ESCs, of which the doubling generation time was 48 hs. CD29 and CD49d were positive;CD71 and CD34 were negative;CKi9, CK15, and P63 were positive. Compared with that of control group, ESCs cultured in chitinous membrane leachate showed slight cell proliferation when diluted to 1:8-1:512 dilutions, but there was no statistically significant difference (P>0.05). The checkerboard-form cell colonies of ESCs could be visualized with naked eyes on the chi-tinous membrane in 2-4 weeks of culture. A multitude of ESCs were seen to grow on fibres under microscope. Conclusions Chitinous membrane may be used as ESCs culture vehicle, and biological compatibility is good.
